The real breakthrough occurred in the 1990s with the commercial release of glyphosate-resistant (GR) crops. These crops allowed the application of glyphosate multiple times in the growing season without the risk of crop injury. Glyphosate was, hitherto, used nonselectively for weed control in vineyards, orchards, rights-of-way, industrial areas, and railroads. It has been deemed as “a once-in-a-century herbicide” [7
] for its broad weed spectrum, reasonable cost, favorable environmental properties, and association with the widely popular GR crops. In susceptible plants, glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimate pathway responsible for the biosynthesis of aromatic amino acids and several secondary metabolites in the phenylpropanoid pathway.
GR maize was introduced in 1998 [5
]. Transformation of maize plants with CP4
species strain) EPSPS
and e35S promoter produced plants with vegetative resistance to glyphosate but reduced male fertility [8
]. Therefore, the first-generation GR maize, trademarked as Roundup Ready®
(RR) trait, GA21 utilized the rice actin 1 promoter driving the gene for a GR form of maize EPSPS (TIPS-EPSPS
]. A new event NK603, with two copies of a slightly modified EPSPS CP4
gene, was developed to improve maize tolerance to glyphosate at both vegetative and reproductive stages, and was commercially released in 2001 in a breeding stack with glufosinate and four insect resistance traits [5
Glufosinate inhibits the enzyme glutamine synthetase (GS), which catalyzes assimilation of ammonia with glutamate to form glutamine [8
]. Glufosinate resistance is due to metabolic inactivation by an acetyltransferase enzyme that catalyzes the acetylation of glufosinate [5
]. Two glufosinate resistance genes, bar
, encode homologous enzymes [9
]. Both genes were isolated from soil microorganisms, pat
from Streptomyces viridochromogenes
from Streptomyces hygroscopicus
]. Glufosinate-resistant maize was commercialized for the first time in 1996 stacked with Bt
insect resistance, as a stand-alone trait in 1997 [6
], and was combined with GR maize as a ‘double stacked trait’ in the mid-2000s.
2,4-D is an auxin herbicide with phytotoxic action limited to broadleaf weed species. However, 2,4-D-resistant maize was developed in tandem with 2,4-D-resistant soybean, trademarked as Enlist™ Weed Control System by Corteva Agriscience (process described in a later section) and deregulated by the US Department of Agriculture (USDA) and an associated low volatility 2,4-D choline formulation registered by EPA for use only in 2,4-D-resistant crops, both in 2014, but not commercially launched in the US until 2018. It is to be noted that 2,4-D had been labeled for use in maize over the past several decades, both as preemergence and postemergence applications. A type of aryloxyalkanoate dioxygenase (AAD) enzyme was identified that provided resistance to 2,4-D as well as a class of ACCase inhibiting herbicides, popularly known as ‘fops’ belonging to the aryloxyphenoxypropionate (AOPP) chemical family, for example, quizalofop [10
]. 2,4-D and the ‘fop’ herbicides possess an identical bond that facilitates their metabolism by a common enzyme. Due to concerns that GR grass weeds would run amuck before commercialization of 2,4-D-resistant crops, 2,4-D-resistant maize was promoted to control GR grass. Another class of ACCase inhibiting herbicides, the ‘dims’ belonging to the cyclohexanedione chemical family, lack the above bond and can be used to manage volunteer 2,4-D-resistant maize. Invariably, most transgenic maize HRCs on the market also carry insect-resistance traits (Bt
trait), which will not be discussed here.
In 1996, GR soybean was the first GR crop to be commercialized. The first generation of GR soybean, event 40-3-2, were the most successful outcome of over-expressing the glyphosate-insensitive CP4 EPSPS
in all tissues using strong, constitutive viral promoters such as e35S or FMV from cauliflower or figwort mosaic viruses, respectively [8
]. The first-generation GR soybean went off patent in 2015, which means individuals can grow them and save seed for re-use as long as the seed has no other trait or varietal patents [12
]. Although the first-generation GR soybean has been phased out of the seed stock (of formerly Monsanto Co., now Bayer Crop Science), some institutions in Missouri and Arkansas have done breeding with this older trait and developed cultivars exhibiting the trait.
The second generation of GR soybean was commercialized in 2009 with a broader launch in 2010 as Roundup Ready 2 Yield®
(RR2Y) by Monsanto Co. [8
]. Several seed companies still sell RR2Y cultivars and they were available in 2019 [12
]. The RR2Y event, MON89788 contained the same CP4 EPSPS
as GTS 40-3-2, but with the gene inserted at a different site in an elite variety “A3244” with a different promoter and regulatory elements to enhance expression in the sensitive tissues [5
Glufosinate-resistant soybean, with the pat
gene and the CaMV 35S promoter, was publicly released for sale in 2009 as a promising tool to combat GR weeds, especially tall water hemp (Amaranthus tuberculatus
Moq. Sauer) and Palmer amaranth (Amaranthus palmeri
S. Watson) [5
]. The glufosinate-resistant trait in soybean has been a good candidate for stacking in other herbicide-resistant soybean cultivars (discussed in a later section).
From the 2019 seed sales season, the glufosinate resistance technology now rests in the hands of BASF Crop Protection, who purchased it from Bayer Crop Science as part of anti-trust remediation [12
]. Glufosinate-resistant crops including soybean have been steadily gaining market share as GR weeds spread across the southern and midwestern US, approaching 20% of the soybean market share. Low seed prices coupled with availability of generic glufosinate herbicides make this technology, labeled as LibertyLink System®
, a viable option for soybean growers and is available on BASF’s Credenz®
soybean platform and other independent seed companies totaling 78 licensees [12
Dicamba-resistant soybean, Roundup Ready 2 Xtend®
(RR2Xtend) from formerly Monsanto Co., now Bayer Crop Science, was deregulated by the Animal and Plant Health Inspection Service (APHIS) of the USDA in 2015. Dicamba monooxygenase (DMO), from the soil bacterium Pseudomonas maltophilia
(strain DI-6), encodes for Rieske nonheme monooxygenase that converts dicamba to 3-6-dichlorosalicylic acid (DCSA) [13
]. The genetically engineered version of the DMO
gene for expression in higher plants used the FLt36 promoter from peanut chlorotic streak virus, a translational enhancer from the tobacco etch virus (TEV), a chloroplast transit peptide–coding region from the pea Rubisco small subunit gene for chloroplast localization of DMO, and a terminator region from the pea Rubisco small subunit gene (rbcS3′
Formulations of dicamba specifically labeled for use in RR2Xtend were not registered until fall 2016. Three dicamba formulations, XtendiMax®
, both containing the diglycolamine (DGA) salt of dicamba, and Engenia®
comprising the BAPMA (N, N-Bis-(aminopropyl) methylamine) salt of dicamba were registered for use in the US by EPA in 2016 until 2018; in 2018 registration was extended until December 2020. In 2017, additional restrictions were implemented toward application of the above formulations which were labeled for use only in dicamba-resistant crops. In 2019, Tavium®
containing dicamba DGA salt plus S
-metolachlor was registered by EPA. In 2016, illegal/off-target/off-label applications of dicamba via formulations other than XtendiMax®
, and Engenia®
were made on dicamba-resistant soybean and cotton (Gossypium hirsutum
L.) in AR, MO, TN, MS, and several other states, resulting in injury to non-dicamba-resistant crops and sensitive flora across the landscape from dicamba drift (volatile/vapor drift and/or physical drift due to droplet movement owing to temperature inversion and other factors). The issue of injury to non-dicamba-resistant crops from dicamba drift was compounded multifold in 2017 when registered dicamba applications in dicamba-resistant soybean were made over large swaths of the cropping area. Dicamba had been labeled for use in maize over the past several decades, both as preemergence and postemergence applications. However, dicamba has not been applied in the middle of the growing season, when temperatures are usually higher than during preplant or early crop growing conditions, or when several sensitive plant species are present, prior to commercialization of dicamba-resistant crops. In the 2017 growing season, a total of 1.44 million ha of dicamba-injured soybean were estimated from 2708 official dicamba-related injury investigations as reported by individual state departments of agriculture and state extension weed scientists in the US (Figure 1
]. It was believed, by several row-crop production practitioners, that the soybean hectarage reported above is a gross underestimation. In 2018, there were fewer complaints of soybean injury compared to 2017, probably, due to more growers planting dicamba-resistant soybean as an insurance against injury, neighboring growers settling disputes off the record, a marked improvement in efficiency of applications, or a combination of more than one of the above reasons. Records of injury from dicamba drift in 2018 and 2019 are available elsewhere in the literature.
The RR2Xtend crop hectarage estimates ranged from 16.2 to 20.2 million ha in 2018 [12
]. The RR2Xtend soybean trait was available from a variety of seed companies via licensing agreements in 2019. It allows growers to spray dicamba and glyphosate postemergence to the crop.
Another group of auxin HRCs are the 2,4-D- resistant crop technologies, developed by formerly Dow AgroSciences and MS Technologies, now managed by Corteva Agriscience. Several species of bacteria possess families of tfdA
genes that are known to produce 2,4-D-metabolizing enzymes [15
]. A tfdA
transgene, originally isolated from the bacterium Alcaligenes eutrophus
], conferred resistance to 2,4-D when expressed in cotton. This gene, independently discovered in bacteria isolated from soil exposed to 2,4-D [18
], increased tolerance in grapes (Vitis vinifera
L.) to 2,4-D by 100-fold.
gene isolated from a gram-negative soil bacteria, Sphingobium herbicidovorans
, codes for a Fe(II) and 2-ketoglutarate-dependent dioxygenase that degrades the alkanoate side chains of both 2,4-D and members of the AOPP class of ACCase inhibitors to a hydroxyl [10
]. Another gene sequence called aad-12
, isolated from Delftia acidovorans
, codes for a 2-ketoglutarate-dependent dioxygenase that inactivates phenoxyacetate auxins such as 2,4-D and pyridinyloxyacetate auxins such as triclopyr or fluroxypyr, but not commercial AOPPs [11
]. The 2,4-D resistance traits were coded DHT1 for maize and DHT2 for soybean [19
2,4-D-resistant soybean, trademarked as Enlist E3™, contain a single molecular stack providing resistance to glufosinate and glyphosate were commercialized in 2019. The original Enlist soybean with 2,4-D and glyphosate resistance was deregulated in 2014 but was not planted due to international import limitations. Another type of Enlist soybean, Enlist Soy + RR2Y, also confers tolerance to these herbicides, but Corteva Agriscience and MS Technologies focused their 2019 commercial offerings on Enlist E3 soybean in the US, Canada, and Brazil after international import restrictions were lifted by China and Philippines in early 2019 [12
]. In 2018, some farmers in Indiana, Illinois, and Ohio planted Enlist E3 soybean within a stewarded closed-loop system based on an agreement between then Dow AgroSciences and Archer Daniels Midland (ADM) Company, as well as seed production acres. Corteva Agriscience is working with more than 100 independent seed companies to broadly license the Enlist E3 soybean trait, which will enable wide availability for 2020 and beyond [20
2,4-D formulations labeled for use with the Enlist Weed Control System include Enlist™ Duo (2,4-D choline salt + dimethylammonium salt of glyphosate) and Enlist™ One (2,4-D choline salt) with COLEX-D™ Technology. 2,4-D products that do not contain COLEX-D™ Technology are not authorized for use in conjunction with Enlist crops.
Herbicides that inhibit the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) represent the last group of commercialized chemical weed control products with a unique mode of action. A new herbicide resistance technology, designated as GT27™ Soybean Performance System, originally developed by Bayer Crop Science and associated entities, confers resistance to glyphosate and a new HPPD-inhibiting herbicide isoxaflutole (ALITE 27, previously Balance Bean) applied preemergence only, pending EPA registration as of July 2019. The trait was commercially available in 2019 as a standalone product offered by some independent seed companies. However, BASF, which now owns the LibertyLink® technology, has made this technology available in 2019 as LibertyLink® GT27™ stack through its Credenz® soybean platform (maturity groups from 0 to 4.5) providing the option to use glyphosate, ALITE 27, and glufosinate. The major risk is that growers will be drawn to applying cheaper generic HPPD inhibitors on the LL GT27 soybean, inviting crop injury.
MGI soybean, carrying resistance to mesotrione, glufosinate, and isoxaflutole, were codeveloped by BASF and Syngenta [21
]. The MGI soybean trait, that received approval from China, is scheduled for commercial launch in 2020, contingent on all regulatory approvals.